The peptide hormone insulin stimulates the uptake and storage of glucose and other nutrients into adipose tissue and skeletal muscle while simultaneously repressing glucose efflux from the liver. Insulin resistance occurs when a normal dose of the hormone is incapable of eliciting these anabolic responses, and the condition is a major contributor to the pathogenesis of type 2 diabetes. Numerous studies suggest that lipid oversupply to these peripheral tissues contributes to the development of insulin resistance, perhaps by promoting the accumulation of fat-derived metabolites capable of inhibiting insulin action. The sphingolipid ceramide has been shown to accumulate in insulin resistant tissues and to inhibit insulin signaling in isolated cells. Moreover, we recently found that endogenous ceramide is both a sufficient and necessary intermediate linking saturated free fatty acids to the inhibition of insulin signaling in cultured myotubes. The studies desribed herein will investigate the contribution of ceramides to the development of insulin resistance in rodents. First, using various strategies to regulate ceramide levels in rodents, including pharmacological inhibitors and a novel knockout mouse that is incapable of making ceramide in most tissues, we will determine the quantitative impact of the sphingolipid in the development of insulin resistance. Second, we will investigate the molecular mechanisms accounting for the antagonistic effects of ceramide on insulin action. And third, we will investigate the regulatory mechanisms controlling ceramide accrual. Information garnered from these experiments could suggest novel therapeutic strategies in the treatment of insulin resistance and type 2 diabetes. [unreadable] [unreadable] [unreadable]